The overall objective is elucidation of the processes of iodothyronine biosynthesis on the molecular level by multidisciplinary approaches. Emphasis of the proposed research is centered on an organic chemical approach using regio- and stereospecifically labeled substrates (3H,14C, 2H, 13C, and 18O at positions on the side chain and ring of tyrosine and its derivatives) 18O2 and (18O) water to delineate the precise molecular mechanism(s) of thyroid hormone biosynthesis. Our immediate objectives include: (1) determination of the fates of hydrogen, carbon, oxygen and nitrogen of N-acetyldiiodotyrosine (NAcDIT) in the coupling process, (2) extension of the coupling reaction to include di- and tripeptides and Dunn's postulated pentapeptide for the T4-site in thyroglobulin, (3) identification of "the lost side-chain" of the coupling, (4) elucidation of the kinetic mechanism and determination of the stoichiometric involvement of oxygen in the coupling reaction, (5) measurement of relative rates of T4, T3, rT3 formation n the model systems, (6) attestation of our hypothesis of the push-pull lyase reaction mechanism involving oxygen, (7) formation of ployiodophenoxyiodothyronines by extended coupling reactions and assessment of the biological activity of these new compounds, and (8) assessment of the catalysis by peroxydase of the coupling reaction. The methods of approach include synthesis of regiospecifically labeled N-acetyliodotyrosines and peptides containing DIT, Tyr, and/or MIT; incubation in a well-defined inorganic medium (no iodoproteins or enzymes); kinetic analysis using radioisotope techniques and an oxygen meter; product identification by nuclear magnetic resonance, high pressure liquid chromatography and other organic chemical techniques; and three-dimensional structure determination by X-ray crystallography.